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Exploring the range of protein flexibility, from a structuralproteomics perspective

Mark Gerstein and Nathaniel Echols

Department of Molecular Biophysics and Biochemistry

Yale University

266 Whitney Ave

New Haven, CT 06520

correspondence: mark.gerstein@yale.edu

Changes in protein conformation play a vital role in biochemical processes, frombiopolymer synthesis to membrane transport. Initial systematizations of protein flexibility,in a database framework, concentrated on the movement of domains and linkers.Movements were described in terms of simple sliding and hinging mechanisms ofindividual secondary structural elements. Recently, the accelerated pace and sophisticationof methods for structural characterization of proteins has allowed high-resolution studiesof increasingly more complex assemblies and conformational changes. New dataemphasizes a breadth of possible structural mechanisms, particularly the ability todrastically alter protein architecture and the native flexibility of many structures.

Introduction

Annotations collected in the Database of Macromolecular Movements(http://molmovdb.org) [1,2] currently include more than 240 distinct protein motions,most of which can be directly visualized based on solved structures [3]. Domain motions ofsingle subunits make up the largest subset, but an increasing number of molecular complexesexhibiting large structural rearrangements have been solved. Initial attempts to classify motionsused a convention of "shear" versus "hinge" movements [4], based on the presence or absence ofa maintained interface between moving parts, and typically focused on movements in singledomains or large fragments. The repertoire of protein conformational changes hasgrownconsiderably, incorporating many cooperative movements of subunits and structural changes at aquaternary level, largely due to improvement in methods for structural characterization of largemolecules. Efforts to computationally model the activityof macromolecular assemblages remainlimited by time constraints, but recent studies have used simulation to investigate the globalconformational changes of immense structures such as the F1-ATPase [5], GroEL[6], and the70S ribosome [7].

Here we summarize a number of recent structural studies that illustrate the importanceand diversity of protein motions, concentrating primarily on several groups of related proteinstructures or mechanisms. Although subtle conformational changes down to the level ofalternating sidechain rotamers are often essential to protein function, our focus is primarily onmore global changes involving significant movement of the protein backbone and interactionsbetween tertiary and quaternary elements. Furthermore, many of these changes may involvemultiple distinct intermediate states or must occur on time scales too large to permit conventionalsimulation. We have especially highlighted proteins that display considerable "plasticity" or"fluidity", in terms of changes in fold, interactions within the cell membrane, or movement in thenative form.

An overview of several new motions in the context of the database is presented in Table 1and Figure 1. With the exception of ATP sulfurylase, each structure listed has only a singlechain exhibiting the described motion (though other subunits may be involved). Althoughidentical methods were used for gathering statistics, the manner of the structural changes varieswidely, and most do not easily fall into one of the pre-existing categories. However, all of thestructures shown have one or more flexible linker regions of multiple residues, from which muchof the displacement is derived, and although there are several cases of shearing helices, mobileinterfaces are not usually maintained within a single chain.

Large-scale remodeling

T7 RNA polymerase.

One of the most dramatic conformational changes observed so faris seen in the elongation-phase structure of T7 RNA pol (Figure 1A). Although movement ofsome type is observed across the polymerase family, most examples involve relatively rigiddomains or superdomains and do not result in changes to the overall architecture of the protein,even in structures with a large overall motion such as the RB69 viral DNA polymerase [8,9].The transition from initiation to elongation in the T7 polymerase requires refolding and massivetranslocation of the N-terminal domain, opening an exit tunnel for the seven-base mRNA strandwhich would otherwise be blocked [10,11]. The remainder of the protein undergoescomparatively little movement. The exact impetus for the rearrangement of the structure andpromoter release is not yet clear, but a further intermediate conformation may be involved. Therelocation of the upstream DNA and the apparent lack of movement by the specificity loop seemto prohibit any direct translation, but it is unclear to what extent the moving domains operate asrigid bodies or whether they must partially unfold.

Mad2.

On a smaller scale, the spindle checkpoint protein Mad2

undergoes arearrangement of similar magnitude involving the transposition of beta strands (Figure 1B).Binding of the small peptide MBP1 disrupts the sheet and causes two of the strands to move tothe opposite side, replaced by MBP1, while a smaller strand at the N-terminus dissociates withthe sheet and instead adopts a helical conformation. Additional data indicates a similar transitionupon binding other proteins not related to MBP1 but known to interact with Mad2 [12,13]. Fewexamples exist of such

rearrangement of a beta sheet; the caspase inhibitor p35 [14] and serpinfamily[15] are the most similar to Mad2, but both involve cleavage and re-insertion of part of thepeptide chain by another protein.

Protein synthesis.

Various ribosome-binding proteins display considerable interdomainflexibility, observed by comparison of apo and ribosome-bound forms or their analogues.Ribosomal translocase has been studied in both prokaryotic and eukaryotic hosts, and a recentpair of structures for the eukaryotic form (EF-2) in native form and bound to a translocationinhibitor (Figure 1C) suggests a large rotation and reorientation of several domains associatedwith ribosome binding [16]. Far more severe, however, is the movement in ribosomal releasefactor 2(RF2) determined by two separate cryo-EM studies of the ribosome. Docking of theisolated crystal structure [17] into the low-resolution EM map requires extension of two domains(Figure 1D), including some alterations in tertiary structure [18,19]. Examination of the crystalstructure strongly supports the closed form as the native state in solution, and not acrystallographic artifact.

Membrane Proteins

The improvement of techniques for structural characterization of membrane proteins hasyielded several examples of structural changes in gating and transport, some involvingconsiderable flexibility within the transmembrane region. An example of receptor function byconformational change was found in the structures of FecA [20], where ligand binding alters theconformation of extracellular loops, transmitting the signal to cytoplasmic proteins. Morecomplex motions, however, are observed in several ionic transport proteins, where bending orshearing in the helical bundle is important.

Potassium channels.

The role of large movements in ion channel gating has been shownby a number of experimental studies [21,22], and MacKinnon and coworkers have recentlyinvestigated the specific structural elements involved at atomic resolution. Comparison ofstructurally related potassium channels KcsA and MthK in the closed and open forms,respectively, illustrated a simple mechanism for gating by bending of the inner helix at aconserved position [23]. A more complicated model for the voltage-gated channel was deducedfrom separate structures of the channel and the voltage-sensing paddles, which move up to 20 Åwithin the membrane and extend arginine residues almost to the solution on either side.Movement of the sensors pulls apart the outer helices to open the channel [24].

P-type ATPases.

Among the largest motions known is exhibited by the Ca2+-ATPaseswitching from calcium-bound to calcium-free states, studied by crystallography and cryo-EM[25-27]. The overall structure of the enzyme is comprised of three independent and relativelyrigid cytoplasmic domains, connected by flexible "stalks" to the transmembrane domaincomprised of ten helices. Release of calcium involves a large rotation and translation of thecytoplasmic domains, accompanied by shearing of six of the transmembrane helices (Figure 1E).Although ATP hydrolysis and calcium transport involve several distinct steps, the transition fromE1 to E2 states appears smooth and can be plausibly approximated without intermediates, unlikethe more severe changes described above. Studies of the structurally homologous Na,K ATPaseusing homology modeling and cryo-EM have indicated a similar mechanism for this protein[28,29].

Ring Complexes

A number of known or suspected motions occur in complexes of identical subunitsarranged in a ring, whose motion is essentially cooperative. The best studied of these are GroEL,whose conformational cycle has been investigated by a host of biophysical techniques includingsimulation, and aspartate transcarbamoylase. Three new completely unrelated structures ofhexameric ATPases have recently been described whose functionality depends on theconformation of the individual subunits.

ATP sulfurylase.

This enzyme catalyzes the incorporation of inorganic sulfur, and isallosterically inhibited by a downstream intermediate inPenicillium. The hexamer consists oftwo stacked rings of three subunits; crystal structures of the R-

and T-states differ mainly by therotation of the C-terminal allosteric domain upon inhibitor binding, which slightly expands thevolume of the overall hexamer. A separate loop movement in the catalytic domain results in amore open active site [30,31].

VirB11.

An ATPase inH. pylori

involved in secretion, VirB11 forms a simple hexamerwhose subunits

adopt multiple conformations in the apo form. The N-terminal domain rotatesaway from the nucleotide-binding site, to varying degrees in each chain, but the structure isstabilized by the interaction of the C-terminal domains. ATP-analogue binding results in a stableconfiguration with the N-

and C-terminal domains closed; hydrolysis does not appear to beresponsible for any further motion, since the ATP and ADP forms have identical conformations.The authors suggest a cycle of ATP binding, hydrolysis,and release which occurs unevenlyamong groups of three subunits [32].

p97/VCP.

A multi-purpose enzyme containing "AAA" ATPase domains, this structureexpands during hydrolysis, based on crystal and cryo-EM structures [33,34]. Disorder of the N-terminal

domain in the cryo-EM maps indicates that the subunits of this structure also haveconsiderable natural flexibility even while assembled into the complete complex, but adopt aspecific conformation during ATP hydrolysis.

Other structures

Several other structures that are not readily classified deserve mention, especially in thecontext of the inherently dynamic complexes described above. As in VirB11, evidence formultiple conformations is frequently found in single crystals, where two or more molecules

inthe asymmetric unit adopt different domain orientations. The two alpha subunits of thetetrameric Acetyl-CoA synthase (Fig 1H) are iron-sulfur binding proteins with three largedomains connected by hinges. In the crystal structure, the exchange of anickel ion for a zincresults in a "closed" configuration of the domains. Although the open form, with two nickelions, appears to be the active state, the role of the conformational change is unclear [35]. Thestructure of ribose-5-phosphate isomerase A

also has two conformations within a crystal, butwithout any change in bound heteroatoms. The mobile domains close around a cleft thought tobe important for substrate binding, apparently due simply to natural flexibility of the tertiarystructure [36].

Recent crystallographic studies that explore the toxic activity of the anthrax bacterium areof particular relevance. Structures of both the lethal factor and the oedema factor exhibit domainmotions in different functional states. The lethal factor, aprotease which attacks signalingpathway kinases in the host cell, displays several small reorientations of elements throughout thestructure (overall RMSD of 1.18) upon binding a target peptide [37]. Far greater flexibility isseen in the oedema factor,an adenyl cyclase, whose activity and structure are altered by bindingof calmodulin. The N-terminal helical domain is displaced and rotated to open a large cleft forcalmodulin. Alteration of the conformation of smaller segments results in activation of

thecyclase [38]. This relatively large motion does not require any particular contortions ofsecondary or tertiary structure, but significant parts of the mobile domain are missing from thefinal model.

Conclusions

Theoretical studies of protein motion have traditionally focused on structures of singlemolecules following a known transition (for instance, domain closure in response to ligandbinding [41]), or on detailed mechanistic and energetic analyses using simulation. Comparisonof multiple structures is limited both by available CPU power and the diversity of tertiaryarrangements, but some trends may nonetheless be seen by a proteomics approach. The degreeof movement in many of the structures examined is striking, particularly in light of the

variety ofmechanisms involved. Along with the repeated observation of mobile domains in crystals, thesestudies indicate the importance of a retaining a large degree of conformational freedom in foldedproteins and reinforce the importance of studying the mechanisms that enable structuralmalleability.

Supplemental Material

Most of the structures discussed for which 3D data is available are listed online athttp://molmovdb.org/molmovdb/cocb, including additional images and animations.

Acknowledgements

The authors would like to thank Thomas Steitz and Whitney Yin for discussions of T7polymerase function, and Michael Barnett for assistance with the database.

(Crystal structure of the elongation phase reveals a massive conformational changeinvolving extensive remodeling of the N-terminal domains. This is one of the most drasticstructural transitions observed.)

(These structures, with and without translocation inhibitor sordarin, indicate a domainmovement associated with the ribosome-bound state, previously suggested by structures of theprokaryotic homolog EF-G.)

(A high-resolution strucure of the E1 state of Ca2+-ATPase, demonstrating considerablereorientation of the cytoplasmic domains accompanied by significant changes in thetransmembrane region. Along with the T7 polymerase and haemagluttinin, one of the largestchanges observed in a single polypeptide chain.)

Quantitative comparison of motions observed by comparison of recent proteinstructures. "Residues" is the consensus length of the protein in the two PDB entries compared,accounting for truncations (only chain A was compared in ATP sulfurylase). RMSD of themobile domains and maximum C--movingparts of the protein fitted, using CNS [40] and the procedure described in [3], respectively.Percentile values are based on comparison to all other motions in the database. Graphicalcomparisons of the different states of each structure are shown in Figure 1.